High molecular weight RNA (35S) isolated from AMV viruses directs the cell-free synthesis of two prominent polypeptides of 180,000 and 76,000 molecular weight. Two-dimensional tryptic peptide analysis of the (35S)-methionine-labeled peptides demonstrate that the 180,000-dalton product is a polyprotein that can account for all the peptides of the AMV virus DNA polymerase. This is direct confirmation of the genomic order of the viral structural genes, placing the polymerase adjacent to the 5'-proximal gag gene of the virus. Furthermore, these findings suggest that the primary polymerase gene product is the beta subunit of the enzyme. These results are discussed in relation to the proposed structural gene map for the avian retroviruses and suggest a model for the in vivo processing of viral polymerase.